Optically active n-hydroxy-alpha-amino acids, amides and their derivatives

ABSTRACT

The invention relates to new pure D- or L-N-hydroxy-alpha-amino acids and the derivatives thereof, including optically active N-hydroxy-phenylglycine amide, which has antibiotic and antitumor activity.

This is a division of application Ser. No. 07/305,903, filed Feb. 3,1989.

The invention relates to a process for the preparation ofN-hydroxy-alpha-amino acids and derivatives thereof by reacting analpha-amino acid derivative with an aromatic aldehyde to form a Schiffbase, oxidizing the Schiff base to an oxaziridine and converting theoxaziridine into the corresponding N-hydroxy-alpha-amino acid derivativeand, if so desired, converting that derivative into the correspondingacid or into a different derivative.

There is an increasing interest in N-hydroxy-alpha-amino acids andderivatives thereof, such as esters, amides, etc., and also peptideswhich contain such compounds as building blocks. Such peptides cannot beprepared by oxidation of an N-H bond in a peptide consisting of one ormore alpha-amino acids. The peptides referred to can only be prepared bycoupling an N-hydroxy-alpha-amino acid derivative to one or morealpha-amino acids.

N-hydroxy-alpha-amino acids and/or derivatives thereof and also peptidesthereof usually have biological activity and mostly antibiotic and/orantitumour activity. E. Buehler and G. B. Brown, J. Org. Chem. 32 (1967)265, state that several N-hydroxyamino acids are components of severalantibiotics that are obtained in microbiological fermentations and theseauthors mention a number of N-hydroxy-alpha-amino acids that areobtained from naturally occurring peptides.

A large number of synthesis reactions are known forN-hydroxy-alpha-amino acids and derivatives thereof. A survey is givenin the article "N-hydroxy-alpha-amino acids in organic chemistry" byHarry C. J. Ottenheym and Jacobus D. M. Herscheid in Chem. Rev. 86(1986) 697-707. This shows that there is a great interest in this typeof compounds. In a previous article, in Synthesis 1980 890, M. W.Tijhuis, J. D. M. Herscheid and H. C.. Ottenheym pointed out that thesynthesis reactions described so far were time-consuming, gave smallyields and were of limited use.

The alpha-carbon atom of alpha-amino acids and derived compounds isalmost always an asymmetrical carbon atom. The only case in which thiscarbon atom is not assymmetrical is if two equal atoms or radicals arebound to it, as is the case in, for example, glycine (amino-aceticacid). In all other cases there are stereoisomers of alpha-amino acids,N-hydroxy-alpha-amino acids and the derivatives thereof.

The aforementioned articles do not or hardly discuss the preparation ofpure or virtually pure stereoisomers of the aforementioned compounds,although this is of great importance, particularly if the compounds areto be used for biological purposes, for example as antibiotics or asagents for inhibiting the growth of tumours. The great importance ofthis was not realized until in the past decennium. Receptors appear toplay a role here.

Among the many synthesis reactions that have been suggested forN-hydroxy-alpha-amino acids or the derivatives thereof there is one thatconsists of the conversion of an ester of an amino acid with an aromaticaldehyde to a Schiff base, oxidation of the Schiff base to anoxaziridine and conversion of the oxaziridine into anN-hydroxy-alpha-amino acid or an ester thereof. This synthesis reactionis known from the article "Oxidation of amino acid esters intoN-hydroxyamino acid derivatives" by T. Polonski and A. Chimiak;Tetrahedron Letters 1974, 2453-2456. However, this gives no or only anincomplete description of the way in which the different reactions arecarried out. Apparently, no attention has been paid to this since thisarticle, which is now 14 years old, in spite of the apparent interest inN-hydroxyamino acids. According to the aforementioned article, theconversion of an ester of an amino acid with an aromatic aldehyde iseffected using the hydrochloride of that ester. For the conversion withan aromatic aldehyde to a Schiff base, the NH₂.HCl group is converted insitu into the NH₂ group and according to the reaction equation this isdone with triethylamine. Other data on the reaction conditions arelacking. Then the Schiff base is oxidized with peracetic acid to form anoxaziridine.

The first, virtually simultaneous descriptions of oxaziridines were byW. D. Emmons in J. Am. Chem. Soc. 78 (1956), 6208 and 79 (1957), 5739and by L. Horner and E. Jurgens in Chem. Ber. 90 (1957), 2184, in whicharticles they are referred to as oxaziranes.

Oxaziridines are highly reactive compounds and Polonski and Chimiak(Loc. cit.) state that the conversion of esters of amino acids withanisaldehyde and oxidation of the resulting Schiff bases results inoxaziridines, which are highly sensitive to acids. When heated withhydrochloric acid, the oxaziridines produce N-hydroxyamino acids. Whenthe hydrochlorides of amino acid esters are used, the yields obtainedare always less than 50% and in many cases they are about 30%. In orderto obtain esters instead of the corresponding acids, the oxaziridinering must be opened under very mild conditions.

Another disadvantage of this process established by the applicant isthat the conversion of an amino acid ester with an aromatic aldehydemust be carried out in complete absence of water, because otherwise theester will be at least partly hydrolyzed to the corresponding acid. Theacid does not react or hardly reacts with the aromatic aldehyde to forma Schiff base. Therefore, the conversion must be effected in an organicsolvent that is free from water. Since water is produced in the reactionof an amino acid ester and an aromatic aldehyde, this water must becontinuously removed from the reaction mixture, for example bydistillation.

It has already been explained above that, in the case of biologicallyactive compounds with possible stereoisomers, it must be possible toprepare the stereoisomer with the desired biological activity as free aspossible from the other possible stereoisomer(s). In some cases it wasfound that one or more other stereoisomers may be very harmful and maylead to extremely undesired side effects. At best, other stereoisomersare not harmful, in which case they constitute undesired ballast in abiologically active compound. It is therefore desirable to be able toobtain N-hydroxy-alpha-amino acids and/or derivatives thereof as pure orvirtually pure stereoisomers. Pure stereoisomers of alpha-amino acidesters are difficult to obtain, unless use is made of a purestereoisomer of an alpha-amino acid.

It has now been found that N-hydroxy-alpha-amino acids can be preparedeasily and with good yields by reacting an alpha-amino acid amide withan aromatic aldehyde, such as benzaldehyde, preferably apara-substituted benzaldehyde, such as anisaldehyde, to thecorresponding Schiff base, oxidizing this Schiff base to thecorresponding oxaziridine and converting the oxaziridine intoN-hydroxy-alpha-amino acid amide. If so desired, theN-hydroxy-alpha-amino acid amide thus obtained can be converted into thecorresponding acid or a different derivative thereof, includingpeptides, such as mono-N-hydroxydipeptides.

More specifically, the present invention relates to compounds with thegeneral formula ##STR1## and the corresponding acids, esters and otherderivatives, as well as to the preparation thereof using compounds withthe general formula ##STR2## in which formulas R₁ represents H, acyclicor cyclic alkyl, whether or not substituted, or aryl, whether or notsubstituted, and R₂ represents H, acyclic or cyclic alkyl, whether ornot substituted, or aryl, whether or not substituted, it beingunderstood that substituents in the groups represented by R₁ and/or R₂are not oxidized under the process conditions or can be protected fromreacting under the process conditions by the introduction of protectivegroups.

If, for example, the alkyl or aryl groups represented by R₁ and/or R₂are substituted by one or more thiol groups or primary or secondaryamino groups, these groups must be protected. The way in which reactivegroups can be protected is well known in the art and is not dealt withany further here.

Preferably, R₂ represents a hydrogen atom. More preferably, the presentinvention relates to N-hydroxy-alpha-amino acid amides and thecorresponding acids, esters and other derivates that are derived fromthe known naturally occurring amino acids, in particular: glycine,alanine, valine, leucine, isoleucine, methionine, phenylalanine,tryptophan, serine, threonine, cysteine, tyrosine, asparagine,glutamine, aspartic acid, glutamic acid, lysine, arginine and histidine.Of these only glycine has a symmetrical alpha-carbon atom. The inventioncomprises both the L-form and the D-form and mixtures of the L- andD-forms of the N-hydroxy-alpha-amino acids and derivatives thereof thatare derived from the other naturally occurring alpha-amino acids. Ofcourse, this also applies to N-hydroxy-alpha amino acids and derivativesthereof that are not derived from the aforementioned naturally occurringamino acids.

The N-hydroxy-alpha-amino acids discussed here are new compounds.Pritzkow and Rosler, Liebigs Ann. Chem. 703 (1967) 66-67 have describedalpha-hydroxylamine-isobutyric acid amide, alpha-hydroxyl-aminecyclohexane carboxylic acid amide and 4-methyl-1-hydroxylaminecyclohexane carboxylic acid amide. The applicant has found that thepreparation method described by Pritzkow and Rosler is of very limiteduse only.

The first step of the process discussed here, the conversion of analpha-amino acid amide with an aromatic aldehyde to form a Schiff base,can be effected in an aqueous medium and results in virtuallyquantitative yields. In this respect the present process differsextremely favourably from the aforementioned process according toPolonski and Chimiak (loc. cit.). The use of an alpha-amino acid amideas reaction component also presents the advantage that alpha-amino acidamides can easily be obtained in the form of pure stereoisomers.Mixtures of stereoisomers of an amino acid amide can be separated byhydrolyzing one of the isomers with an aminopeptidase, after which theremaining amide isomer can be separated from the acid formed. By thenreconverting the hydrolyzed isomer into the amide again, bothstereoisomers of the amide can be obtained in a pure form. Such aprocess is described in, for example, U.S. Pat. No. 3,971,700 andNL-A-75.13551. In this way, it is much easier to prepare purestereoisomers of alpha-amino acid amides than to prepare purestereoisomers of alpha-amino acid esters, in which case use must be madeof the pure steroisomers of the acids.

Many processes are known for the preparation of individual stereoisomersof alpha-amino acids, for example conversion of a mixture ofstereoisomer of such an acid with a stereoisomer of a base, selectivecrystallization of the salt thus formed and conversion of a stereoisomerof the salt into the acid. If the acid must be esterified, losses willoccur in the process and there will mostly also be a certain degree ofracemization. Because of this, the preparation of stereoisomers ofalpha-amino acid esters is a much more time-consuming process than thepreparation of stereoisomers of alpha-amino acid amides and, moreover,the yield of esters in the overall conversion reaction is much lowerthan that of amides. Alpha-amino acid amides can be prepared with verygood yields from alpha-amino nitriles and in the manner mentioned abovethe alpha-amino acid amides can then easily, and with high yields, beseparated into the individual stereoisomers. The conversion of astereoisomer of an alpha-amino acid amide into the correspondingN-hydroxy-alpha-amino acid amide according to the present invention doesnot only appear to result in very high yields, as already mentionedabove, but it also takes place without any racemization.

This the applicant discovered by converting a stereoisomer of analpha-amino acid amide into an N-hydroxy-alpha-amino-acid amideaccording to the present process and then reducing the latter back to analpha-amino acid amide with hydrogen and the aid of a Pd/C catalyst. Thepossible stereoisomers of the alpha-amino acid amides andN-hydroxy-alpha-amino acid amides investigated are enantiomers. Theoptical activity is a measure of the purity.

The alpha-amino acid amide obtained in the reduction reaction appearedto present the same specific rotation as the alpha-amino acid amide usedas starting material. From this it can be concluded that no racemizationwhatsoever took place. This is a great advantage, because it is knownthat racemization occurs in very many conversions, even in conversionsthat do not involve the asymmetric carbon atom itself.

The Schiff base that is formed in the reaction of an alpha-amino-acidamide and an aromatic aldehyde is very insoluble in water. If thisconversion is effected in an aqueous medium, the Schiff baseprecipitates and can easily be recovered by filtration. As alreadymentioned above, the Schiff base yields are practically quantitative.

The Schiff base is then oxidized into an oxaziridine. The oxidation ofthe Schiff base must be effected in such a manner that it does notproceed any further once oxaziridine is obtained. Any person skilled inthe art can easily determine how to effect the oxidation while ensuringthat it does not proceed beyond the oxaziridines. Organic peracids, suchas peracetic acid, perbenzoic acid, chloroperbenzoic acid,monoperphthalic acid, etc., appeared to be particularly suitable forthis purpose. Highly suitable is m-chloroperbenzoic acid. This oxidationcan be carried out in a suitable manner by dissolving the Schiff base inan organic solvent that is free from water, such as dichloromethane, anether, which may also by cyclic such as dioxane, tetrahydrofuran, etc.

Using m-chloroperbenzoic acid presents an advantage, because itdissolves in a solvent like dichloromethane, in which the Schiff base isalso soluble. The oxidation, which is preferably effected at roomtemperature, results in oxaziridine that is soluble in the organicsolvent, and m-chlorobenzoic acid, which is insoluble in, for example,dichloromethane and therefore precipitates, after which it can beremoved by filtration.

Without being isolated from the solution, the oxaziridine can then beconverted into the corresponding N-hydroxy-alpha-amino acid amide byacid hydrolysis. This hydrolysis must be carried out carefully to avoidhydrolysis of the amide group. The reason for this is that thehydroxyamino acid that is formed in further hydrolysis is not verystable and partial decaboxylation takes place in an acid medium, whichmay cause considerable losses. The acid hydrolysis must be carried outin a moderately acid medium. The hydrolysis may, for example, beeffected in a suitable manner with hydroxylamine hydrochloride (NH₂OH.HCl), a weakly acidically reacting compound, the pH of a 0.1 msolution of which amounts to 3.4 at 25° C. Other acid compounds with asimilar acidity may also be used.

The hydroxylamine hydrochloride that is preferably to be used is notvery soluble in apolar solvents such as dichloromethane, but is verysoluble in alcohols, in particular in methanol (at 25° C., 17.5 g/100 g)and therefore the hydrolysis is preferably effected in an alcoholic,more preferably a methanolic medium. The oxaziridine solution indichloromethane can be evaporated and the residue introduced into, forexample, methanol, but it is also possible to add, for example, methanolto the solution in dichloromethane. The hydrolysis proceeds faster in analcoholic medium than in an alcohol-dichloromethane mixture andtherefore evaporation and introduction into an alcohol are preferable.

The solution of the oxaziridine in an alcohol or a mixture of an alcoholand a non-polar or slightly polar solvent, such as dichloromethane, towhich, for example, hydroxylamine hydrochloride has been added, isstirred at ambient temperature until the oxaziridine has beenhydrolyzed, which usually takes a few hours. Then an apolar solvent,such as ether, is added to precipitate the N-hydroxy-alpha-amino acidamide as HCl salt, which can then be recovered by separation, forexample by filtration.

The embodiments described above of the preparation ofN-hydroxy-alpha-amino acid amides were only described for a betterunderstanding of the invention. It will, however, be clear that theinvention is not limited thereto and that the invention comprises anyembodiment according to which the steps of the process according to theinvention, being: (1) conversion of alpha-amino acid amide and aromaticaldehyde into a Schiff base, (2) oxidation of the Schiff base intooxaziridine, (3) conversion of oxaziridine into N-hydroxy-alpha-aminoacid amide, can be carried out.

The N-hydroxy-alpha-amino acid amides according to the invention can beconverted into the corresponding acids, esters and other derivatives.

The hydrolysis of the amides discussed here to the correspondingN-hydroxy-alpha-amino acids can, with particular advantage, be effectedenzymatically, with the aid of an amido hydrolase, for example asdescribed in NL-A-84.03093. When this enzymatic hydrolysis is completed,the remaining enzymes are removed, for example by centrifugation, afterwhich sufficiently pure N-hydroxy-alpha-amino acid can be recovered, forexample by evaporating the solution. As compared with this, thehydrolysis of the amides discussed here with the aid of acidsconstitutes a considerable burden on the environment. Another importantadvantage of enzymatic hydrolysis is that it can be effected at a pHbetween 6 and 10. In an acid medium N-hydroxy-alpha-amino acids are onlymoderately stable, much less stable than the corresponding alpha-aminoacids at the same pH. In an acid medium N-hydroxy-alpha-amino acids areeasily decarboxylized, particularly when heating is applied, as isnecessary in acid hydrolysis.

The N-hydroxy-alpha-amino acids according to the invention can beconverted into esters or other derivatives in a known manner.

The N-hydroxy-alpha-amino acids or derivatives thereof according to thepresent invention can also be used for the preparation ofN-hydroxypeptides, a group of compounds that is also discussed in thearticle by Ottenheym and Herscheid (loc. cit.)

The invention is further elucidated with the following examples.

These examples describe ways in which to prepare stereoisomers that arenot or hardly contaminated by one or more other stereoisomers. Ofcourse, the invention is not at all limited to these examples andcomprises both other stereoisomers and mixtures of stereoisomers. Theinvention is not limited by the examples in any other way either.

EXAMPLE I

a) 12.5 g (0.11 mol) of D-valine amide was dissolved to a 10 wt %solution in water with stirring and gentle heating to 40° C. The pH wasset to at least 11 by adding 1N KOH. Then 16.5 g (0.12 mol) ofanisaldehyde (4-methoxybenzaldehyde) was added dropwise, with stirring,over a period of about 15 minutes. The mixture was allowed to cool toroom temperature and the stirring was continued for about 2 hours. Thenthe precipitated Schiff base of D-valine amide and anisaldehyde wasremoved by filtration, washed with water to remove anisaldehyde remainsand dried. The yield was almost quantitative (98%).

b) 23.4 g (0.1 mol) of the Schiff base of D-valine and anisaldehydeprepared according to Ia) was dissolved, with stirring, in 130 ml of drydichloromethane. The solution was cooled to 5° C. or lower in an icebath. Then 20.6 g (0.12 mol) of meta-chloroperbenzoic acid was added,with stirring, to the solution cooled to 5° C. or lower. The stirringwas continued and the temperature was allowed to rise to roomtemperature, after which the stirring was continued for 4 more hours.After about 0.5 hour's stirring at room temperature the mixture becameturbid as a result of the crystallization of meta-chlorobenzoic acid.After the reaction, in which all the meta-chlorobenzoic acid formedcrystallized, the reaction mixture was filtered and the filteredsolution was evaporated until dry.

c) The evaporation residue obtained according to Ib) was dissolved in150 ml of methanol. With stirring, 7.0 g (0.10 mol) of hydroxylaminehydrochloride was slowly added to this solution, a small portion at atime, after which the stirring was continued for 3 more hours at roomtemperature. The reaction mixture thus obtained was then added dropwiseto 1 l of diethyl ether. A white precipitate of D-N-hydroxyvalineamide.HCl was formed, which was removed by filtration and dried. Yield:12.8 g (76%).

[alpha]²⁰ _(D) =-73°. (c=1, H₂ O).

d) 8.4 g of the D-N-hydroxyvaline amide.HCl was dissolved in 100 ml ofmethanol. The solution was transferred to a Parr apparatus along with a5% Pd on carbon catalyst (5 mol % Pd with respect to the hydroxyvalineamide) and reduced with hydrogen. After hydrogenation the Pd/catalystwas removed by filtration and the solution in methanol was evaporated.The specific rotation of the hydrogenated product appeared to be thesame as that of D-valine amide.HCl, namely -28.3°. This shows that thepreparation of D-N-hydroxyvaline amide.HCl from D-valine amide.HClaccording to b) takes place without racemization.

EXAMPLE II

a) The Schiff base of D-phenyglycine amide and anisaldehyde was preparedin the same manner as in example I.

b) 26.8 g (0.1 mol) of the Schiff base of D-phenylglycine amide andanisaldehyde was dissolved in 115 ml of dry dichloromethane. Thesolution was cooled to 0° C. in an ice bath and then 24.3 g (0.12 mol)of meta-chlorobenzoic acid was added, with stirring. With stirring, thetemperature was allowed to rise to room temperature, after which thestirring was continued for 4 more hours. After approximately 1/4 hour'sstirring at room temperature the mixture became turbid as a result ofthe crystallization of m-chlorobenzoic acid. After the reaction, inwhich all the m-chlorobenzoic acid formed had crystallized, the reactionmixture was filtered, after which the filtered solution was evaporateduntil dry.

c) The evaporation residue obtained according to IIb) was dissolved in150 ml of methanol. With stirring, 7.0 g (0.10 mol) of hydroxylaminehydrochloride was slowly added to this solution, a small portion at atime, after which the mixture was stirred for 5 more hours at roomtemperature. With stirring, the reaction mixture thus obtained was thenadded dropwise to 2 l of diethylether. A white precipitate ofD-N-hydroxyphenylglycine amide.HCl was formed, which was removed byfiltration and dried. Yield: 17.6 g (85%).

[alpha]²⁰ _(D) =-107.5° (c=1, MeOH).

d) 6.3 g of D-N-hydroxyphenylglycine amide.HCl was dissolved in 10 ml ofwater, after which 2.12 g of sodium carbonate, free from water, wasadded to the solution, with stirring. A precipitate was formed, whichwas removed by filtration, washed twice with 5 ml of water and thendried.

Yield: 4.5 g of D-N-hydroxyphenylglycine amide.H₂ O.

[alpha]²⁰ _(D) =-57.6° (c=1, MeOH)

NMR (DMSO): delta, dpm: 4.32 (H alpha), 6.10 (N-H), 7.54 (OH), 7.14/7.42(CONH₂), 7.2-7.4 (phenyl), J (H alpha, NH)=8 Hz, J (NH, OH)=2 Hz.

e) The preparation processes described in sections a), b) and c) of thisexample were repeated using L-phenylglycine amide instead ofD-phenylglycine amide. This resulted in the same yield ofL-N-hydroxyphenylglycine amide.HCl with [alpha]²⁰ _(D) =+107.5° (c=1,MeOH).

EXAMPLE III

In the same way as in example II the Schiff bases of D-phenylalanineamide and anisaldehyde and of L-phenylalanine amide and anisaldehydewere prepared in two experiments. Of each of these bases a solution wasprepared of 21.0 g (0.074 mol) in 140 ml of dichloromethane. Thesolutions were cooled to 0° C. in an ice bath, after which 16.1 g (0.09mol) of meta-chloroperbenzoic acid was added to each solution, withstirring, after which the cooling was stopped to allow the temperatureto rise to room temperature. The solutions, in which after 10 minutesalready a precipitate started to form, were stirred for 4.5 hours, afterwhich the precipitate was removed by filtration. The solutions weredried by evaporation and the evaporation residues were each dissolved in100 ml of methanol, after which 6.3 g of hydroxylamine hydrochloride wasadded to each solution. After 7 hours' stirring at room temperature, 2 lof diethyl ether was slowly added to each solution. The resultingprecipitates were removed by filtration and dried. Yields: 13.4 g (86%)of D-N-hydroxyphenylalanine amide.HCl and 13.0 g (83%) ofL-N-hydroxyphenylalanine amide.HCl.

By dissolving 6.7 g of these compounds in 10 ml of water and adding 2.12g of sodium carbonate, free from water, 5.4 g ofD-N-hydroxyphenylalanine amide.H₂ O and the corresponding L-compoundwere prepared, which had specific rotations of -4.1° (D) and +4.3° (L),respectively. (c=1, MeOH).

NMR (DMSO): delta, dpm: 2.62 (H beta), 2.77 (H beta), 3.45 (H alpha),5.59 (N--H), 7.41 (OH), 7.01/7.15 (CONH₂), 7.1-7.3 (phenyl), J (H beta1, H beta 2)=14 Hz, J (H beta 1, H alpha)=8 Hz, J (H beta 2, Halpha)=5.5 Hz, J (H alpha, NH)=8.5 Hz, J (NH, OH)=3 Hz.

EXAMPLES IV-VIII

In the same way as described in the previous examples,L-N-hydroxyleucine amide.HCl was prepared from L-leucine amide (exampleIV) with a yield of 65% and the corresponding N-hydroxy compounds wereprepared from racemic mixtures of phenylglycine amide (example V),phenylalanine amide (example VI) and valine amide (example VII).

In the same manner D,L-alpha-methyl-alpha-N-hydroxy-valine amide.HCl wasprepared from the Schiff base of anisaldehyde and alpha-methylvalineamide (racemic mixture) (example VIII).

We claim:
 1. The D- or L- stereo-isomer of a compound with the formula##STR3## in substantially optically pure form, in which R₁ represents H,acyclic or cyclic alkyl or carbocyclic aryl and R₂ represents H, acyclicor cyclic alkyl or carbocyclic aryl, with the following provisoregarding combinations of R₁ and R₂ : R₁ and R₂ are not, respectively, Hand H; nor CH₃ and CH₃ ; nor H and CH₃ ; nor CH₃ and H.
 2. A compoundaccording to claim 1, in which one of the symbols R₁ and R₂ representsH.
 3. L-N-hydroxy-phenylglycine amide, in substantially optically pureform.
 4. D-N-hydroxy-phenylglycine amide, in substantially opticallypure form.